The objective of this proposal is a detailed elucidation of mechanism and transition state structure in enzyme catalyzed hydrogen abstraction reactions. Enzymes which both activate hydrogen and are characterized by a broad substrate specificity will be studied. The combined application of structure reactivity correlations and isotope effects facilitates the isolation of chemical bond making (breaking) steps in enzyme reactions, and provides insigt into fundamental questions in enzymology concerning (1) the mode of hydrogen activation (H:vs H ion vs H) in enzyme catalyzed redox reactions, (2) the participation of active site residues in acid-base catalysis; (3) the changes in charge and bond hydridization which accompany the formation of the enzyme transition state complex; and (4) the contribution of hydrophobic, steric and electronic factors to substrate binding and the relationship of these factors to catalysis. Primary hydrogen isotope effects and structure reactivity correlations in the yeast alcohol dehydrogenase reaction indicate a rate limiting C-H cleavage step; secondary isotope effects will be measured as probes of changes in bond order at reacting bonds in the enzyme transition state. Experiments will be carried out to distinguish between two highly dissimilar modes of hydrogen activation (H:vs H) in this reaction. In order to compare transition state structure among alcohol dehydrogenases from divergent evolutionary sources, the C-H cleavage step of a bacterial dehydrogenase will be isolated kinetically and characterized. The stereochemistry of the plasma amine oxidase catalyzed exchange of protons from C-2 of phenylethylamines will be determined. This exchange reaction will provide a facile route for the synthesis of a series of isotopically labelled, para-substituted phenylethylamines, which will be used in a study of substituent and isotope effects in the dopamine-beta-hydroxylase (DBH) reaction. In conjunction with kinetic studies, chemically reactive analogs of dopamine and fumarate will be tested as alkylating agents of the substrate and activator binding sites of DBH.